ABSTRACT
The radiosynthesis of [N-methyl-(11)C]Org 34850 as a potential brain glucocorticoid receptor (GR)-binding radiotracer is described. The radiosynthesis was accomplished via N-methylation of the corresponding desmethyl precursor with [(11)C]methyl triflate in a remotely controlled synthesis module to give the desired compound in a radiochemical yield of 23+/-5% (decay-corrected, based upon [(11)C]CO(2)) at a specific activity of 47+/-12 GBq/micromol (n=15) at the end-of-synthesis (EOS). The radiochemical purity after semi-preparative HPLC purification exceeded 95%. The total synthesis time was 35-40 min after end-of-bombardment (EOB). The radiotracer is rapidly metabolized in rat plasma leading to the formation of two more hydrophilic metabolites as the major metabolites. Radiopharmacological evaluation involving biodistribution and small animal PET imaging in normal Wistar rats showed that the compound [N-methyl-(11)C]Org 34850 is not able to sufficiently penetrate the blood-brain barrier. Therefore, compound [N-methyl-(11)C]Org 34850 seems not to be a suitable PET radiotracer for imaging rat brain GRs. However, involvement of Pgp or species differences requires further clarification to establish whether the radiotracer [N-methyl-(11)C]Org 34850 may still represent a suitable candidate for imaging GRs in humans.
Subject(s)
Carbon Radioisotopes , Radiopharmaceuticals/chemical synthesis , Receptors, Glucocorticoid/metabolism , Steroids/chemical synthesis , Sulfones/chemical synthesis , Animals , Blood-Brain Barrier/metabolism , Humans , Methylation , Positron-Emission Tomography , Radiopharmaceuticals/metabolism , Radiopharmaceuticals/pharmacokinetics , Rats , Rats, Wistar , Steroids/metabolism , Steroids/pharmacokinetics , Sulfones/metabolism , Sulfones/pharmacokineticsABSTRACT
The luteinizing hormone (LH) receptor plays a pivotal role in reproduction. The high-molecular-weight (HMW) human chorionic gonadotropin (hCG) and LH are the endogenous ligands of this receptor and bind to its large N terminus. The present study characterizes the binding of a new low-molecular-weight (LMW) radioligand, [(3)H]5-amino-2-methylsulfanyl-4-[3-(2-morpholin-4-yl-acetylamino)-phenyl]-thieno[2,3-d]pyrimidine-6-carboxylic acid tert-butylamide (Org 43553), at the LH receptor. Equilibrium saturation and displacement assays were developed and optimized. Specific binding of [(3)H]Org 43553 to CHO-K1 cell membranes expressing the human LH receptor and a cAMP response element-luciferase reporter gene was saturable with a K(D) value of 2.4 +/- 0.4 nM and a B(max) value of 1.6 +/- 0.2 pmol/mg protein. Affinities of five LMW analogs of Org 43553 were determined. All displaced the radioligand competitively, with K(i) values ranging from 3.3 to 100 nM. Finally, the potency of these compounds in a cAMP-induced luciferase assay was also determined. There was a high correlation between affinity and potency (r = 0.99; P < 0.0001) of these compounds. In the search for LMW ligands, which bind allosterically to the seven-transmembrane domain of the LH receptor, a HMW radioligand (e.g., (125)I-hCG) is not suitable as it is not displaced by a LMW compound. Therefore, [(3)H]Org 43553, a new radioligand with good binding properties, allows screening for new LMW ligands that mimic the action of the endogenous hormone at the LH receptor.
Subject(s)
Pyrimidines/chemistry , Pyrimidines/pharmacology , Radioligand Assay , Receptors, LH/agonists , Receptors, LH/metabolism , Thiophenes/chemistry , Thiophenes/pharmacology , Allosteric Regulation/physiology , Animals , CHO Cells , Cricetinae , Cricetulus , Dose-Response Relationship, Drug , Humans , Luteinizing Hormone/analogs & derivatives , Luteinizing Hormone/metabolism , Luteinizing Hormone/pharmacology , Molecular Weight , Pyrimidines/metabolism , Radioligand Assay/methods , Thiophenes/metabolism , Tritium/metabolismABSTRACT
A steroid 15beta-hydroxylating whole-cell solvent tolerant biocatalyst was constructed by expressing the Bacillus megaterium steroid hydroxylase CYP106A2 in the solvent tolerant Pseudomonas putida S12. Testosterone hydroxylation was improved by a factor 16 by co-expressing Fer, a putative Fe-S protein from Bacillus subtilis. In addition, the specificity for 15beta-hydroxylation was improved by mutating threonine residue 248 of CYP106A2 into valine. These new insights provide the basis for an optimized whole-cell steroid-hydroxylating biocatalyst that can be applied with an organic solvent phase.
Subject(s)
Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Pseudomonas putida/genetics , Solvents/pharmacology , Testosterone/metabolism , Gene Expression Regulation, Bacterial , Hydroxylation , Models, Biological , Mutagenesis, Site-Directed , Organisms, Genetically Modified , Substrate SpecificityABSTRACT
Taxine B (3), isolated from the dried needles of Taxus baccata, was converted into six novel 7-deoxypaclitaxel analogs, 20, 21a,b, and 23-25, that have structural changes at C1, C2, and C4. A method for the introduction of the benzoyl function at C2, via a benzylidene acetal at C1-C2, will be revealed. All compounds showed very little or no measurable cytotoxic activity against some well-characterized human tumor cell lines, probably due to the nonacylated hydroxyl group at C4.
